Metabolomics-based study of logarithmic and stationary phases of promastigotes in Leishmania major by [1]H NMR spectroscopy

Background: Cutaneous leishmaniasis is one of the most important parasitic diseases in humans. In this disease, one of the responsible organisms is Leishmania major, which is transmitted by sandfly vector. There are specific differences in biochemical profiles and metabolite pathways in logarithmic and stationary phases of Leishmania parasites. In the present study, [1]H NMR spectroscopy was used to examine the metabolites outliers in the logarithmic and stationary phases of promastigotes in L. major to enlighten more about the transmission mechanism in metacyclogenesis of L. major

Methods: Promastigote was cultured, logarithmic and stationary phases were separated by the peanut agglutinin, and cell metabolites were extracted. [1]H NMR spectroscopy was applied, and outliers were analyzed using principal component analysis

Results: The most altered metabolites in stationary and logarithmic phases were limited to citraconic acid, isopropylmalic acid, L-leucine, ornithine, caprylic acid, capric acid, and acetic acid

Conclusion: [1]H NMR spectroscopy could play an important role in the characterization of metabolites in biochemical pathways during a metacyclogenesis process. These metabolites and their pathways can help in exploiting a transmission mechanism in metacyclogenesis, and outcoming data might be used in the metabolic network reconstruction of L. major modeling

The Route of Leishmania tropica Infection Determines Disease Outcome and Protection against Leishmania major in BALB/c Mice

Leishmania tropica is one of the causative agents of leishmaniasis in humans. Routes of infection have been reported to be an important variable for some species of Leishmania parasites. The role of this variable is not clear for L. tropica infection. The aim of this study was to explore the effects of route of L. tropica infection on the disease outcome and immunologic parameters in BALB/c mice. Two routes were used; subcutaneous in the footpad and intradermal in the ear. Mice were challenged by Leishmani major, after establishment of the L. tropica infection, to evaluate the level of protective immunity. Immune responses were assayed at week 1 and week 4 after challenge. The subcutaneous route in the footpad in comparison to the intradermal route in the ear induced significantly more protective immunity against L. major challenge, including higher delayed-type hypersensitivity responses, more rapid lesion resolution, lower parasite loads, and lower levels of IL-10. Our data showed that the route of infection in BALB/c model of L. tropica infection is an important variable and should be considered in developing an appropriate experimental model for L. tropica infections.

Expression and single-step purification of GRA8 antigen of toxoplasma gondii in Escherichia coli

Diagnosis of Toxoplasma gondii [T.gondii] infection is of great medical importance especially for pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several tToxoplasma antigens, including dense granule antigens [GRAs] has a great potential as diagnostic reagents. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags. In the present study, we produced truncated GRA8 [GRA8], excluded from the signal peptide and C-terminal transmembrane domain, with a short fusion tag in Escherichia coli. [E.coli]. GRA8 was purified using an optimized single-step Iimmobilized Mmetal ion Aaffinity Cchromatography [IMAC]. The purity and yield of GRA8 was highest at pH= 9.25. At this pH, 13.6 mg of GRA8 was obtained with the purity of 97.97%. Immunogenicity of the protein was evaluated in Western blot analysis showing the serum sample from a rabbit immunized with GRA8 recognized a single antigen of T. gondii tachyzoite at the expected molecular weight of native GRA8. To diagnosis acute tToxoplasma infection in pregnant women, an indirect immunoglobulin M [IgM] enzyme-linked immunosorbent assay [ELISA] was developed using GRA8 resulting in a test specificity and sensitivity of 97.1% and 60.6%, respectively. These results demonstrated that immunogenic GRA8 can be produced in fusion with a short tag and purified near to homogeneity using an optimized IMAC. GRA8-IgM-ELISA was useful for detection of acute toxoplasma infection

Immunotherapy with imiquimod increases the efficacy of glucantime therapy of leishmania major infection

Leishmaniasis is a complex disease which presents as visceral, cutaneous and mucocutaneous forms. The current treatment options for this infection are very limited and the immunological state of the host appears to play an important role in the efficacy of the treatment. Immunostimulation through immune response activating agents such as Imiquimod is another rational approach for this purpose. We assessed the efficacy of immunotherapy with Imiquimod alone or combined with Glucantime for treatment of Leishmania major infection in BALB/c mice. Treatment efficacy was monitored by determination of thickness and parasite load of infected foot-pad of mice. The footpad thickness revealed that treatment with Imiquimod plus Glucantime has the highest efficacy. The results were confirmed by parasite load of infected footpad. Our data shows that treatment of Leishmania major infection in BALB/c mice by the combined Imiquimod and Glucantime is more efficient than each drug alone. The combination of Imiquimod with chemotherapy may offer a way for more efficient treatment of leishmaniasis

Detection of anti-platelet glycoprotein antibodies Using MAIPA method

AITP mostly occur in children accompanied by variable clinical sings including petechiae, purpura, ecchymosis and severe bleeding. This study has determined and characterized the anti-platelet glycoproteins in children with ITP. The aim of this study was to determinate anti-platelet glycoproteins [GPs] using MAIPA method. During 18 months 38 children with clinical signs of AITP were studied in Mofid children hospital. To determine anti-platelet antibodies by ELISA technique, washed O negative platelets were used as a source of platelet antigens. MAIPA method was used to detect antibodies against individual platelet membrane glycoprotein. The anti-platelet antibodies level above mean+ 3SD of control group was assumed as positive. The results indicated that the platelet count ranges was between 2×109/L and 95×109/L. 63.5 % out of 38 patients were anti-platelet antibodies positive with ELISA method. The correlation between the above patients with anti-platelet antibody positive and clinical signs was 0.4. Results for determination of antibody against platelet GPIIb/IIIa, GPIb/IX and GPIa/IIa using MAIPA method were 44%, 51% and 25% respectively. In conclusion the preference of MAIPA method is the detection of very small amount of antibody. Since MAIPA is the specific method for the detection of antibody against glycoprotein antigens, it has the advantage of differentiating immune and non-immune thrombocytopenia

Detection of Helicobacter pylori infection by imported IgG ELISA kits in comparison with Iranian home made kit

Helicobacter pylori [Hp] is a gram negative, spiral shaped bacterium which colonizes the gastric mucosa and induces gastroduodenal complications varying from mild gastritis with no clinical complications to peptic ulcer diseases and even gastric malignancies. The rate of Hp infection is 30-50% in developed countries whereas it has been rated up to 80% of the adult population in developing countries like Iran. Hp infection can be detected by various diagnostic methods. Culturing biopsy specimens and Rapid Urease Test [RUT] are the most common and reliable tests which can manifest Hp infection through proper sampling but these methods are invasive ones due to the need for endoscopy procedure in isolation of biopsy specimen. Application of serological assays are being increasingly used for epidemiological studies and detecting systemic immune responses toward past Hp infection. ELISA assays are the most popular techniques particularly in cases with no previous treatment. In this study we tested three imported IgG ELISA kits which are available for clinical diagnostics in detecting host sero-reactivity to Hp infection and compared them with a home made IgG ELISA kit. Histology and RUT were used as the gold standard tests for determination of Hp positive vs. Hp negative subjects using biopsy specimens from antrum. Sensitivity, specificity, accuracy and other required criteria were evaluated for each ELISA kit. According to the results the original criteria [Sensitivity and specificity] for each kit were as follows: BIOHIT [41.6%, 100%], Trinity [100%, 86.6%], Pishtaz [100%, 86.6%], Home made [100%, 92.6%]. Evaluation of these different IgG ELISA kits originating from different parts of the world and cross comparison of the results indicated that the cut off values should be refined for user country in order to obtain the highest sensitivity and specificity. These differences can be due to the vast geographic heterogeneity among Hp antigens. Furthermore, this study showed that home made ELISA kit can be substituted for imported ELISA kits due to its valid serological criteria

Evaluation of a new anti-HIV11/2 ELISA-HIV 1/2 REC diagnostic kit based on E. coli derived soluble recombinant proteins: Experience of an international study

Development of a new enzyme-linked immunosorbent assay [ELISA] for screening human blood serum and plasma for antibodies to human immunodeficiency virus type 1 [HIV1] and type 2 [HIV2] as HIV1/2REC ELISA diagnostic kit based on E. coli derived soluble recombinant proteins. Polypeptides corresponding to HIV1 gp41 and HIV2 gp36 immunodominant regions and HIV1 gag were expressed in E. coli in fusion with thioredoxin [Trx] to obtain a highly purified [>98%] soluble refolded proteins, which was used as solid phase antigens for ELISA. The sensitivity and specificity of anti-HIV1/2 antibody detection were evaluated with representative panels of positive and negative sera. Positive panels included HIV1-positive Western-blot [WB]-confirmed specimens collected in Iran, Russia, and Uganda. Commercially available HIV1 and HIV2 seroconversion low titer and performance panels were also used. Negative panel was collected from random volunteer blood donors, risk group members, HCV-infected patients and individuals with non-HIV related conditions potentially influencing test results. The sensitivity of antibody detection with new kit was determined to be 100%. Specificity was determined to be 99.82%. It was shown than thioredoxin [Trx] did not change the immunodominant epitopes of HIV. These fusion proteins are recognized by human native antibodies. In addition, thioredoxin [Trx] would help natural refolding of HIV proteins by E. coli. These characteristics of the new assay are comparable to those of majority of FDA-licensed and officially approved European diagnostic kits, which are currently available in the United States and Europe